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Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Arabinogalactan proteins (AGPs) are important plant proteoglycans involved in many development processes. In roots, AGPs occur in the cell wall of root cells and root cap-derived cells as well as in the secreted mucilage. Detection, localization , and quantification techniques are therefore essential to unravel the AGP diversity of structures and functions. This chapter details root-adapted immunocytochemical methods using monoclonal antibodies, and a collection of biochemical analysis protocols using β-D-glucosyl Yariv reagent for comprehensive AGP characterization.

International audience; Root border cells (BCs) and their associated secretions form a protective structure termed the root extracellular trap (RET) that plays a major role in root interactions with soil borne microorganisms. In this study, we investigated the release and morphology of BCs of Glycine max using light and cryo-scanning electron microscopy (SEM). We also examined the occurrence of cell-wall glycomolecules in BCs and secreted mucilage using immunofluorescence microscopy in conjunction with anti-glycan antibodies. Our data show that root tips released three populations of BCs defined as spherical, intermediate and elongated cells. The mechanism of shedding seemed to be cell morphotype-specific. The data also show that mucilage contained pectin, cellulose, extracellular DNA, histones and two hemicellulosic polysaccharides, xyloglucan and heteromannan. The latter has never been reported previously in any plant root secretions. Both hemicellulosic polysaccharides formed a dense fibrillary network embedding BCs and holding them together within the mucilage. Finally, we investigated the effect of the RET on the interactions of root with the pathogenic oomycete Phytophthora parasitica early during infection. Our findings reveal that the RET prevented zoospores from colonizing root tips by blocking their entry into root tissues and inducing their lysis.

Arabinogalactan proteins (AGPs) are important plant proteoglycans involved in many development processes. In roots, AGPs occur in the cell wall of root cells and root cap-derived cells as well as in the secreted mucilage. Detection, localization , and quantification techniques are therefore essential to unravel the AGP diversity of structures and functions. This chapter details root-adapted immunocytochemical methods using monoclonal antibodies, and a collection of biochemical analysis protocols using β-D-glucosyl Yariv reagent for comprehensive AGP characterization.

International audience; Pea (Pisum sativum) root cap releases a large number of living border cells that secrete abundant mucilage into the extracellular medium. Mucilage contains a complex mixture of polysaccharides, proteins and secondary metabolites important for its structure and function in defense. Unlike xyloglucan and cellulose, pectin and arabinogalactan proteins have been investigated in pea root and shown to be major components of border cell walls and mucilage. In this study, we investigated the occurrence of xyloglucan and cellulose in pea border cells and mucilage using cytochemical staining, immunocytochemistry and laser scanning confocal microscopy. Our data show that i) unlike cellulose, xyloglucan is highly present in the released mucilage as a dense fibrillary network enclosing border cells and ii) that xyloglucan and cellulose form molecular cross-bridges that tether cells and maintain them attached together. These findings suggest that secreted xyloglucan is essential for mucilage strengthening and border cell attachment and functioning.

International audience; The root cap releases cells that produce massive amounts of mucilage containing polysaccharides, proteoglycans, extracellular DNA (exDNA) and a variety of antimicrobial compounds. The released cells – known as border cells or border‐like cells – and mucilage secretions form networks that are defined as root extracellular traps (RETs). RETs are important players in root immunity. In animals, phagocytes are some of the most abundant white blood cells in circulation and are very important for immunity. These cells combat pathogens through multiple defence mechanisms, including the release of exDNA‐containing extracellular traps (ETs). Traps of neutrophil origin are abbreviated herein as NETs. Similar to phagocytes, plant root cap‐originating cells actively contribute to frontline defence against pathogens. RETs and NETs are thus components of the plant and animal immune systems, respectively, that exhibit similar compositional and functional properties. Herein, we describe and discuss the formation, molecular composition and functional similarities of these similar but different extracellular traps.

International audience; Extensins are cell wall glycoproteins, belonging to the Hydroxyproline-Rich GlycoProtein (HRGP) family, which are involved in many biological functions, including plant growth and defence. Several reviews have described the involvement of HRGPs in plant immunity but little focus has been given specifically to cell wall extensins. Yet, a large set of recently published data indicates that extensins play an important role in plant protection, especially in root-microbe interactions. Here, we summarize the current knowledge on this topic and discuss the importance of extensins in root defence. We first provide an overview of the distribution of extensin epitopes recognised by different monoclonal antibodies among plants and discuss the relevance of some of these epitopes as markers of the root defence response. We also highlight the implication of extensins in different types of plant interactions elicited by either pathogenic or beneficial microorganisms. We then present and discuss the specific importance of extensins in root secretions as these glycoproteins are not only found in the cell walls but are also released into the root mucilage. Finally, we propose a model to illustrate the impact of cell wall extensin on root secretions.

International audience